Development of a fluorescence stereomicroscope and observation of Bong-Han corpuscles inside blood vessels

A fluorescence stereomicroscope system was developed in order to observe in situ the distribution of nuclei in intravascular Bong-Han ducts and corpuscle tissues by injecting acridine orange, which stained specifically nuclei. Intravascular Bong-Han corpuscles, connected with Bong-Han ducts could be detected in the aortas of rats, mice, and rabbits.     

Structural and biochemical bases for the inhibition of autophagy and apoptosis by viral BCL-2 of murine gamma-herpesvirus 68

All gammaherpesviruses express homologues of antiapoptotic B-cell lymphoma-2 (BCL-2) to counter the clearance of infected cells by host antiviral defense machineries. To gain insights into the action mechanisms of these viral BCL-2 proteins, we carried out structural and biochemical analyses on the interactions of M11, a viral BCL-2 of murine gamma-herpesvirus 68, with a fragment of proautophagic Beclin1 and BCL-2 homology 3 (BH3) domain-containing peptides derived from an array of proapoptotic BCL-2 family proteins. Mainly through hydrophobic interactions, M11 bound the BH3-like domain of Beclin1 with a dissociation constant of 40 nanomole, a markedly tighter affinity compared to the 1.7 micromolar binding affinity between cellular BCL-2 and Beclin1. Consistently, M11 inhibited autophagy more efficiently than BCL-2 in NIH3T3 cells. M11 also interacted tightly with a BH3 domain peptide of BAK and those of the upstream BH3-only proteins BIM, BID, BMF, PUMA, and Noxa, but weakly with that of BAX. These results collectively suggest that M11 potently inhibits Beclin1 in addition to broadly neutralizing the proapoptotic BCL-2 family in a similar but distinctive way from cellular BCL-2, and that the Beclin1-mediated autophagy may be a main target of the virus.     

Antioxidant activities of ethanol extracts from seeds in fresh Bokbunja (Rubus coreanus Miq.) and wine processing waste

The antioxidant potential of ethanol extracts from defatted Bokbunja seed wastes generated during wine processing were estimated by radical scavenging abilities (DPPH(*), H(2)O(2), and O(2)(*-)), retardation of lipid oxidation, and iron ion-chelating characteristics. For comparison, ethanol extracts from seeds of fresh ripe Bokbunja fruits were also used. The ethanol extracts from the wine seed waste always showed higher scavenging activities against DPPH(*), H(2)O(2), and O(2)(*-) than those from the fresh seeds. The oxidation of linoleic acid in dimethylsulfoxide at 105 degrees C revealed that the kinetic behavior clearly obeyed pseudo-zero-order regardless of the linoleic acid concentration. The Fe(II)-chelating capacity was determined by the Freundlich isotherm. The results showed high potential and favorability of the two extracts for Fe(II) chelation. The Freundlich chelation capacities (mg(1-1/n)L(1/n)/g) of both ethanol extracts from seeds of fresh Bokbunja and wine processing wastes, tannic acid, and proanthocyanidin from Pinus radiata bark were 100, 224, 260, and 307, respectively. The Fe(III)-chelating properties of the ethanol extracts were considered to be deeply associated with its 3',4',5'-trihydroxyl (galloyl) group as with tannic acid.     

The influence of curvature on membrane domains

An interdependence between local curvature and domain formation has been observed in both cell and model membranes. An implication of this observation is that domain formation in model membranes may be modulated by membrane curvature. In this paper, small-angle neutron scattering (SANS) is used to examine the influence of membrane curvature (i.e., vesicle size) on the formation of membrane domains. It is found that, although vesicle size and polydispersity are not significantly altered by the formation of membrane domains, the area fraction occupied by domains depends on the overall vesicle size. In particular, increasing membrane curvature (i.e., decreasing vesicle size) results in increased area fractions of membrane domains.     

Recombinant antibodies recognize conformation-dependent epitopes of the leucine zipper of misfolding-prone myocilin

Recombinant antibodies with well-characterized epitopes and known conformational specificities are critical reagents to support robust interpretation and reproducibility of immunoassays across biomedical research. For myocilin, a protein prone to misfolding that is associated with glaucoma and an emerging player in other human diseases, currently available antibodies are unable to differentiate among the numerous disease-associated protein states. This fundamentally constrains efforts to understand the connection between myocilin structure, function, and disease. To address this concern, we used protein engineering methods to develop new recombinant antibodies that detect the N-terminal leucine zipper structural domain of myocilin and that are cross-reactive for human and mouse myocilin. After harvesting spleens from immunized mice and in vitro library panning, we identified two antibodies, 2A4 and 1G12. 2A4 specifically recognizes a folded epitope while 1G12 recognizes a range of conformations. We matured antibody 2A4 for improved biophysical properties, resulting in variant 2H2. In a human IgG1 format, 2A4, 1G12, and 2H2 immunoprecipitate full-length folded myocilin present in the spent media of human trabecular meshwork (TM) cells, and 2H2 can visualize myocilin in fixed human TM cells using fluorescence microscopy. These new antibodies should find broad application in glaucoma and other research across multiple species platforms.     

Inflammatory events in hippocampal slice cultures prime neuronal susceptibility to excitotoxic injury: a crucial role of P2X7 receptor-mediated IL-1beta release

We investigated the consequences of transient application of specific stimuli mimicking inflammation to hippocampal tissue on microglia activation and neuronal cell vulnerability to a subsequent excitotoxic insult. Two-week-old organotypic hippocampal slice cultures, from 7-day-old C57BL/6 donor mice, were exposed for 3 h to lipopolysaccharide (LPS; 10 ng/mL) followed by 3 h co-incubation with 1 mM ATP, or 100 microM 2'3'-O-(4-benzoyl-benzoyl) adenosine 5'-triphosphate triethylammonium, a selective P2X(7) receptor agonist. These treatments in combination, but not individually, induced a pronounced activation and apoptotic-like death of macrophage antigen-1 (MAC-1)-positive microglia associated with a massive release of interleukin (IL)-1beta exceeding that induced by LPS alone. Antagonists of P2X(7) receptors prevented these effects. Transient pre-exposure of slice cultures to a combination of LPS and P2X(7) receptor agonists, but not either one or the other alone, significantly exacerbated CA3 pyramidal cell loss induced by subsequent 12 h exposure to 8 microM alpha-amino-3-hydroxy-5-methyl-4-isoxazole propinate (AMPA). Potentiation of AMPA toxicity was prevented when IL-1beta production or its receptor signaling were blocked by an inhibitor of interleukin-converting-enzyme or IL-1 receptor antagonist during application of LPS + ATP. The same treatments did not prevent microglia apoptosis-like death. These findings show that transient exposure to specific pro-inflammatory stimuli in brain tissue can prime neuronal susceptibility to a subsequent excitotoxic insult. P2X(7) receptor stimulation, and the consequent IL-1beta release, is mandatory for exacerbation of neuronal loss. These mechanisms may contribute to determine cell death/survival in acute and chronic neurodegenerative conditions associated with inflammatory events.     

Effect of codon optimization on the enhancement of the β-carotene contents in rice endosperm

A β-carotene is the most well-known dietary source as provitamin A carotenoids. Among β-carotene-producing Golden Rice varieties, PAC (Psy:2A:CrtI) rice has been previously developed using a bicistronic recombinant gene that linked the Capsicum Psy and Pantoea CrtI genes by a viral 2A sequence. To enhance β-carotene content by improving this PAC gene, its codon was optimized for rice plants (Oryza sativa L.) by minimizing the codon bias between the transgene donor and the host rice and was then artificially synthesized as stPAC (stPsy:2A:stCrtI) gene. The GC content (58.7 from 50.9%) and codon adaptation index (0.85 from 0.77) of the stPAC gene were increased relative to the original PAC gene with 76% DNA identity. Among 67 T₁ seeds of stPAC transformants showing positive correlations between transgene copy numbers (up to three) and carotenoid contents, three stPAC lines with a single intact copy were chosen to minimize unintended insertional effects and compared to the representative line of the PAC transgene with respect to their codon optimization effects. Translation levels were stably increased in all three stPAC lines (3.0-, 2.5-, 2.9-fold). Moreover, a greater intensity of the yellow color of stPAC seeds was correlated with enhanced levels of β-carotene (4-fold, 2.37 μg/g) as well as total carotenoid (2.9-fold, 3.50 μg/g) relative to PAC seeds, suggesting a β-branch preference for the stPAC gene. As a result, the codon optimization of the transgene might be an effective tool in genetic engineering for crop improvement as proven at the enhanced levels of translation and carotenoid production.     

Synergic Interface Optimization with Green Solvent Engineering in Mixed Perovskite Solar Cells

Organic–inorganic hybrid halide perovskite solar cells (PSCs) have recently drawn enormous attentions due to their impressive performance (>22%) and low temperature solution processability (<150 °C). Current solution process involves application of a large amount of toxic solvents, such as chlorobenzene, which is heavily employed in both the perovskite layer and the hole transport layer (HTL) deposition. Herein, this study employs green solvent of ethyl acetate for engineering efficient perovskite and HTL layers, which enables a synergic interface (perovskite/HTL) optimization. A champion efficiency of 19.43% is obtained for small cells (0.16 cm² with mask) and over 14% for large size modules (5 × 5 cm²). The PSCs prepared from the green solvent engineering demonstrate superior performance on both efficiency and stability over their chlorobenzene counterparts. These enhancements are ascribed to the in situ inhibition on carrier recombination induced by interfacial defects during the solution processing, which enables about 2/3 reduction of calculated recombination rate. Thus, the green solvent route shows the great potential toward environmental‐friendly manufacturing.     

Protein particulate retention and microorganism recovery for rapid detection of Salmonella

The rapid detection of Salmonella in ground meat requires that living microorganisms be brought to levels detectable by PCR, immunoassays, or similar techniques within 8 h. Previously, we employed microfiltration using hollow fiber membranes to rapidly process and concentrate viable bacteria in food extracts through a combination of enzyme treatment and prefiltration in order to prevent blockage or fouling of the hollow fiber membranes. However, scanning electron microscopy and particle size analysis of enzyme hydrolysates showed that enzyme treatment followed by filtration caused submicron particles to form and be trapped within the prefiltration media, which in turn, retained about 80% of the bacteria. Filtering prior to enzyme treatment resulted in formation of a filter cake consisting of protein particles retained on the surface of the filter, while facilitating passage of the much smaller microorganisms through the filter, separating them from particulates. Subsequent enzyme treatment of the filtrate resulted in an extract that was microfiltered in less than an hour, while concentrating viable microorganisms in the extract by 500×. An inoculum of Salmonella enterica cells into turkey burger containing of 1–20 CFU/mL, consisting of spiked cells plus cells already present in the turkey burger sample, was rapidly brought to levels detectable by conventional PCR and BAX^® PCR assays. The entire procedure from sample processing to detection of Salmonella enterica was achieved in less than 8 h. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:687–695, 2017     

Cytological observation of two environmental genic male-sterile lines of rice.

We report here two environmental genic male-sterilities (EGMS) in rice. These two EGMS rice lines, thermo-sensitive genic male-sterility (TGMS) and photoperiod-sensitive genic male-sterility (PGMS), are controlled by temperature and photoperiod, respectively, in determining their male-sterility. Male-sterility of the TGMS and PGMS was found to be induced when they were grown at 32 degrees C/26 degrees C (day/night) with 14 h daylight, while they were fertile at 26 degrees C/20 degrees C (day/night) with 10 h daylight in a growth chamber. We also examined their anther structures under a light microscope. The light microscopic observation revealed that the EGMS lines showed a complete pollen abortion at the sterile growth condition while they produced normal fertile pollens at the fertile growth condition.